Lecture 7: Protein Sorting and Transport Pt. 2

where are most lipids synthesized?

ER

major membrane lipids

• phospholipids

• glycolipids

• cholesterol

the liver

very rich in smooth ER, contains detoxifying enzymes to metabolize lipid-soluble components

basic components of the membrane

phospholipids

location of enzymes to synthesizes phospholipids

cytoplasmic side of the smooth ER membrane

flippase

flips the phospholipids through the hydrophobic tail regions

KDEL

ER retrieval signal → binds to KDEL receptor and transports from Golgi back to ER

KDEL amino acid sequence

Lys-Asp-Glu-Leu

proteins with ER export signals

will exit from ER to Golgi

topology of secretory pathway

ER lumen side becomes extracellular side of the plasma membrane

vesicular transport from ER to Golgi

• transport vesicles carrying proteins and lipids bud from ER exit sites

• fuse to form vesicle at ER-Golgi intermediate compartment (ERGIC)

• then transport to Golgi

Golgi structure

a stack of flattened cisternae

how vesicles enter/exit Golgi

fusion with the cis (convex) face, then exit trans (concave) face

N-linked oligosaccharides formation location

ER

N-linked oligosaccharides in Golgi

modified in Golgi by a sequence reaction

lipids synthesized in Golgi

• sphingomyelin

• glycolipids

function of lipids synthesized in Golgi

cell recognition and signaling

how are lysosomal proteins targeted/marked?

phosphorylation of mannose

signal patch

how lysosomal proteins are recognized, it is a 3D conformation of the folded protein

where do endosomes mature?

lysosomes

COPII-coated vesicles- direction

ER → ERGIC → Golgi

COPI-coated vesicles- direction

Golgi → ERGIC → ER (backwards)

Clatherin-coated vesicles- direction

both directions

small G protein families involved in vesicle formation

• ARF1

• SAR1

ARF1

ADP ribosylation factors, form COPI and Clatherin-coated vesicles

ARF-GTP

active form, recruits GGA

GGA

binds to cargo bound receptor, recruits AP1

AP1

binds to Clathrin to assemble vesicle

ARF-GDP

inactive form

SAR1

forms COPII coated vesicles

SNARE proteins

v SNARE and t SNARE mediate the fusion of the vesicle membrane and the target membrane

SNARE

soluble NSF attachment protein receptor

v SNARE

on vesicle

t SNARE

on target

what is required to disassemble the SNARE complexes?

energy

exocyst

mediates tethering and localization of transport vesicles to correct location at apical membrane before fusion, involved in exocytosis

exocytosis

vesicles fusing with plasma membrane and releasing their contents outside the cell

endocytosis

capturing a substance or particle from outside the cell by engulfing it with the cell membrane, bringing it into the cell

Rab11 function

in exocytosis: trans Golgi network to plasma membrane

Rab5 function

in endocytosis: plasma membrane to early endosome

Rab7 function

in endocytosis: early endosome to late endosome

Rab GTP-binding proteins to know

• Rab11

• Rab5

• Rab7

Rab

small GTPases that switch between GDP inactive and GTP active forms, involved in vesicular trafficking

GDI

GDP dissociation inhibitor, keeps Rab-GDP bound

GDI-displacement factor

displaces Rab-GDP from GDI

Rab GEF

activates Rab-GDP to Rab-GTP

Rab GAP

deactivates Rab-GTP to Rab-GDP

Griscelli syndrome

caused by mutations in gene encoding Rab27a, causes hypopigmentation due to abnormal transport of melanosomes

lysosome

digestive system in the cell

Gauchers disease

a lysosome storage disease due to failure to hydrolyze glycolipids in lysosome

pH in lysosome → will acid hydrolases work here?

5, yes

pH in cytosol → will acid hydrolases work here?

7, no

proton pump in lysosomes

uses ATP, keeps pH low

steps of targeting proteins to lysosome

• signal patch on lysosomal proteins

• modification of N-linked sugar with mannose-6-phosphate

• transport to lysosome